Traditional ChIP Method

Chromatin IP from Yeast Whole Cell Extracts

Day 1

Grow 5ml overnight.

Day 2

* Dilute cells to OD600= 0.2 in ml.
* Grow until OD600= 0.8-1.0
* Crosslink cultures by adding ml 37% formaldehyde to give final conc of 1%. Incubate for 15min at RT on nutator.
* Quench reaction by adding ml 2.5M glycine (final conc 125mM).
* Transfer cells to 50ml Falcon tubes, spin down 5min, 3000rpm 6C
* Discard media and wash twice by adding ml ice cold 1xPBS, vortex, spin again 5min, 3000rpm, 6C. Asperate liquid.
* Resuspend in ul lysis buffer (add ul protease inhibitors and ul 50mM PMSF) and transfer to 1.5ml siliconized eppendorfs.
* Add equal volume glass beads (about 500ul 0.45-0.52mm, acid wash with dilute nitric acid before use). Lyse cells in Eppendorf shaker for 45-60 min at 4C.
* Spin briefly, invert tubes, puncture bottom and top of tube with red hot 25G1 needle, collect lysate into frest tube 1-2min spin. Place on ice.
* Shear chromatin to average size of 500bp. Sonicate 3x 10sec, with 45 sec total interval on ice = Program 2, 4 Output.
* Centrifuge 10min 4C eppendorf centrifuge and collect supernatant (crude cell lysate). Final volume lysate = ul. Cell lysate can be stored at -80C.
* Setup IPs overnight with 0.5-5ul antibody on nutator at 4C.

Day 3

* Add 25ul 50% slurry of protein A sepharose beads (washed as for [[IP]]) incubate 4C for 2 hours on nutator.
* Pellet beads at 3000 rpm in bench centrifuge 30 sec. Can save supernatant from IP for Westerns.
* Wash beads with 2x 5min RT with 500ul lysis buffer. Can transfer to filter tubes for washes.
* Wash beads with 1x 5min RT with 500ul [[deoxycholate buffer]].
* Wash beads with 1x 5min RT with 500ul TE. Don't use filter tubes for final wash, resuspend in TE and transfer to normal tubes.
* Elute fragments from beads 65C in 50ul [[elution buffer]] for 15mins.
* Spin down and transfer supernatant to new tube.
* Reverse cross-links by incubating at 65C in 50ul [[elution buffer]] overnight.
* For Input: Thaw 20ul frozen cell extract, add 50ul TES, incubate 65C o/n.

Day 4

* Spin 3000g, remove supernatant to new tube to remove beads
* Add 50ul RT TE, and 100ug (5ul) proteinase K, incubate 2-3hr at 55C.
* Add 100ul TE.
* Add 200ul Phenol/chloroform/isoamylalcohol, shake, spin 5min max.
* Remove supernatant to fresh tube, add 100ul TE to phenol tube, spin
* Add 1ul glycogen, 30ul Sodium Acetate, 1ml ice-cold EtOH.
* Incubate -20C 10min - 2hrs
* Spin max, 4C, 30mins
* Add 1ml ice-cold 70% EtOH, spin, 15mins, 4C
* Air dry 10mins
* Resuspend DNA in 50ul TE.
* Store -20C
* For Input: RNase treat half the sample (1ul 10ug 1hr 37C), run on 0.8% agarose gel.

Day 5

* Setup PCRs.

Deoxycholate Buffer

10mM Tris pH8.0 400ul 1M
0.25M LiCl 10ml 1M
0.5% NP-40 800ul 25%
0.5% Sodium doxycholate 2ml 10%
1mM EDTA 80ul 500mM
H2O 25.52ml